Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories : production of human alpha-galactosidase A
Unzueta Elorza, Ugutz (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Vázquez Lima, Felícitas (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Accardi, Giulia (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Mendoza, Rosa (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Toledo-Rubio, Verónica (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Giuliani, Maria (Università degli Studi di Palermo. Dipartimento di Scienze Biochimiche)
Sannino, Filomena (Università degli Studi di Palermo. Dipartimento di Scienze Biochimiche)
Parrilli, Ermenegilda (Università degli Studi di Palermo. Dipartimento di Scienze Biochimiche)
Abasolo, Ibane (Hospital Universitari Vall d'Hebron)
Schwartz, Simon (Hospital Universitari Vall d'Hebron)
Villaverde Corrales, Antonio (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Corchero Nieto, José Luis (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Ferrer-Miralles, Neus (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Tutino, Maria Luisa (Università degli Studi di Palermo. Dipartimento di Scienze Biochimiche)

Data:	2015
Resum:	Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.
Ajuts:	Ministerio de Economía y Competitividad IT2009-0021 Ministerio de Economía y Competitividad EUI2008-03610 Agència de Gestió d'Ajuts Universitaris i de Recerca 2009/SGR-108
Nota:	This work was supported by ERANET-IB08-007 project from the European Union and its linked national project EUI2008- 03610 to AV. We also appreciate the support from EME2007-08 to NFM from Universitat Autonoma de Barcelona, from Antartide 2010 to MLT and EP, from MIUR Azioni Integrate Italia-Spagna 2010 Prot. IT10LECLM9 to MLT, from MINECO (IT2009-0021) to AV and LT, from AGAUR (2009SGR-108) to AV. AV is also supported by The Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN, Spain), an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. PS has received predoctoral fellowship from ISCIII, and AV has been distinguished with an ICREA ACADEMIA award (Catalonia, Spain).
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Llengua:	Anglès
Document:	Article ; recerca ; Versió acceptada per publicar
Matèria:	Recombinant protein ; Expression systems ; Escherichia coli ; Pseudoalteromonas haloplanktis TAC125 ; Human alpha-galactosidase A ; Fabry's disease
Publicat a:	Applied microbiology and biotechnology, Vol 99, Issue 14 (July 2015) , p. 5863-5874, ISSN 0175-7598

DOI: 10.1007/s00253-014-6328-9

Post-print 18 p, 135.9 KB

Material electrònic 8 p, 1.1 MB

Figures 6 p, 382.1 KB

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